Characterization of functionally active subribosomal particles from Thermus aquaticus

Peptidyl transferase activity of,Thermus aquaticus ribosomes is resistant t o the removal of a significant number of ribosomal proteins by protease dig estion, SDS, and phenol extraction. To define the upper limit for the numbe r of macromolecular components required for peptidyl transferase, particles obtained by extraction of T. aquaticus large ribosomal subunits were isola ted and their RNA and protein composition was characterized. Active subribo somal particles contained both 23S and 5S rRNA associated with notable amou nts of eight ribosomal proteins. N-terminal sequencing of the proteins iden tified them as L2, L3, L13, L15, L17, L18, L21, and L22. Ribosomal protein L4, which previously was thought to be essential for the reconstitution of particles active: in peptide bond formation, was not found. These findings; together with the results of previous reconstitution experiments, reduce t he number of possible essential macromolecular components of the peptidyl t ransferase center to 23S rRNA and ribosomal proteins L2 and L3, Complete re moval of ribosomal proteins from T. aquaticus rRNA resulted in loss of tert iary folding of the particles and inactivation of peptidyl transferase. The accessibility of proteins in active subribosomal particles to proteinase h ydrolysis was increased significantly after RNase treatment. These results and the observation that 50S ribosomal subunits exhibited much higher resis tance to SDS extraction than 30S subunits are compatible with a proposed st ructural organization of the 50S subunit involving an RNA "cage" surroundin g a core of a subset of ribosomal proteins.